1. Field of the Invention
The present invention concerns the sector of microbiological analyses.
2. Description of Related Art Including Information Disclosed Under 37 CFR 1.97 and 37 CFR 1.98.
In medicine, in order to combat a resistant germ, it is often necessary to administer one or two antibiotics to a patient at the same time, for which reason it is firstly essential to perform a synergy test in order to verify their compatibility and their associated effect.
The synergy test of two antibiotics performed on a bacterial culture medium is, in fact, essential for the purpose of verifying:                an antagonistic effect of the two antibiotics to be administered to the patient, in the sense that the two antibiotic molecules combined together produce a lesser effect than they would produce if considered individually;—a synergistic effect of the two antibiotics to be administered to the patient, in the sense that the two antibiotic molecules combined together produce a greater effect than the sum of the single effects of the two molecules;        an additive effect of the two antibiotics to be administered to the patient, in the sense that the two antibiotic molecules combined together produce an effect that it is equal to the sum of the single effects of the two molecules;        an indifferent effect of the two antibiotics to be administered to the patient which occurs when the two antibiotic molecules, even if combined together, do not produce an improved effect compared to what they would have produced if considered individually.        
At the present state of the art, there are different methods for testing for synergy between antibiotic molecules: time-kill in broth macrodilution1, checkerboard in broth microdilution2, substitution of the antimicrobial gradient strips on microbial culture through agar diffusion′, crossing of antimicrobial gradient strips on microbial culture through agar diffusion4.    1, 2, 4 Comparison of three different in-vitro methods of detecting synergy: time-kill, checkerboard, and Etest. R L White, D S Burgess, M Manduru and J A Bosso Antimicrob. Agents Chemother. 1996, 40(8): 1914.    1, 2, 3 Comparison of Etest, chequerboard dilution and time-kill studies for the detection of synergy or antagonism between antifungal agents tested against Candida species. Lewis, R., Diekema, D., Messer, S., Pfaller, M. and Klepser, M. (2002). JAC. 49: 345-351.
The determination of the minimum inhibitory concentration (MIC) of an antimicrobial agent against a microorganism through the use of graduated strips impregnated with antimicrobial gradient consists of depositing a strip, made of a porous or non porous material on a microbial population on an agar culture medium. The antibiotic strip releases the antibiotic according to the predefined gradient, and after an incubation period of 18 hours or more, an elliptical inhibition area, symmetrical and centred along the strip, can be observed.
The MIC value, expressed in μg/mL, is read at the intersection point between the lower edge of the inhibition ellipse and the strip.
The synergy test of two antimicrobial agents through the use of the gradient strip with the crossing method of the strips on microbial culture medium firstly involves the determination of the MIC for each antibiotic; then, the strips of the two antibiotics are placed perpendicularly on the bacterial culture. The two strips must cross at the point corresponding to the MIC values of the two individual antimicrobial agents previously determined. After incubation, the new MIC of each antibiotic is evaluated and the combined effect of the same is calculated using the following algorithm5, 6:
Defined:
MICA=MIC of antibiotic A
MICB=MIC of antibiotic B
MICAB=MIC of antibiotic A in presence of antibiotic B;
MICBA=MIC of antibiotic B in presence of antibiotic A.
The FIC (Fractional Inhibitory Concentration index) is calculated:FIC index=MICAB/MICA+MICBA/MICB
SynergyFIC <0.5AdditivityFIC >0.5 and <1.0IndifferenceFIC >1 and <4AntagonismFIC >4    5 Comparison of techniques for measurement of in vitro antibiotic synergism. Norden, C. W., Wentzel, H., Keleti, E. J. Infect. Dis. 140:629-633, 1979.    6 Correlations between methods for measurement of synergy. Berenbaum, M. C. 1980. J. Infect. Dis. 142:476-480.
The accuracy of this synergy test method depends on the correct crossing of the two antimicrobial gradient strips which must be perfectly perpendicular. The performance of this test is susceptible to errors by the operator, who could accidentally fail to position the strips at an angle of 90° or fail to pinpoint the correct crossing point during manual depositing of the second strip on top of the first, which would result in a test to be repeated or in obtaining unreliable results.
The intersection of the strips at 90° is the condition required for the correct evaluation of the inhibition rings and the reading of the value of the MIC on the graduated strip and the consequent calculation of the above-described algorithm.
The depositing of the second strip at the desired crossing point is difficult and susceptible to error because it is common practice in laboratories to perform this operation manually by lab forceps, attempting to achieve a millimetric positioning on the exact point of the graduated strip that corresponds to the MIC point, exposing the operator to natural imprecision. Once the antimicrobial strip has come into contact with the bacterial culture it can no longer be removed nor repositioned inasmuch as the process of diffusion of the antibiotic is immediate when it comes into contact with the agar.